This relates to the fields of genetics, and more particularly relates to site-directed mutagenesis of a gene of interest.
Gene Therapy
Gene therapy is the introduction into a cell of an entire replacement copy of a defective gene to treat human, animal and plant genetic disorders. The introduced gene, via genetic recombination, replaces the endogenous gene. This approach requires complex delivery systems to introduce the replacement gene into the cell, such as genetically engineered viruses, or viral vectors.
Gene therapy is being used on an experimental basis to treat well known genetic disorders of humans such as retinoblastoma and cystic fibrosis. However, in vivo efficiency is low due to the limited number of recombination events actually resulting in replacement of the defective gene.
Triple-Stranded DNA
Since the initial observation of triple-stranded DNA many years ago by Felsenfeld et al., J. Am. Chem. Soc. 79:2023 (1957), oligonucleotide-directed triple helix formation has emerged as a valuable tool in molecular biology. Current knowledge suggests that oligonucleotides can bind as third strands of DNA in a sequence specific manner in the major groove in homopurine/homopyrimidine stretches in duplex DNA. In one motif, a homopyrimidine oligonucleotide binds in a direction parallel to the purine strand in the duplex, as described by Moser and Dervan, Science 238:645 (1987), Praseuth et al., Proc. Natl. Acad. Sci. USA 85:1349 (1988), and Mergny et al., Biochemistry 30:9791 (1991). In the alternate purine motif, a homopurine strand binds anti-parallel to the purine strand, as described by Beal and Dervan, Science 251:1360 (1991). The specificity of triplex formation arises from base triplets (AAT and GGC in the purine motif) formed by hydrogen bonding; mismatches destabilize the triple helix, as described by Mergny et al., Biochemistry 30:9791 (1991) and Beal and Dervan, Nuc. Acids Res. 11:2773 (1992).
Triplex forming oligonucleotides have been found useful for several molecular biology techniques. For example, triplex forming oligonucleotides designed to bind to sites in gene promoters have been used to block DNA binding proteins and to block transcription both in vitro and in vivo. (Maher et al., Science 245:725 (1989), Orson et al., Nucleic Acids Res. 19:3435 (1991), Postal et al., Proc. Natl. Acad. Sci. USA 88:8227 (1991), Cooney et al., Science 241:456 (1988), Young et al., Proc. Natl. Acad. Sci. USA 88:10023 (1991), Maher et al., Biochemistry 31:70 (1992), Duval-Valentin et al., Proc. Natl. Acad. Sci. USA 89:504 (1992), Blume et al., Nucleic Acids Res. 20:1777 (1992), Durland et al., Biochemistry 30:9246 (1991), Grigoriev et al., J. of Biological Chem. 267:3389 (1992), and Takasugi et al., Proc. Natl. Acad. Sci. USA 88:5602 (1991)). Site specific cleavage of DNA has been achieved by using triplex forming oligonucleotides linked to reactive moieties such as EDTA-Fe(II) or by using triplex forming oligonucleotides in conjunction with DNA modifying enzymes (Perrouault et al., Nature 344:358 (1990), Francois et al., Proc. Natl. Acad. Sci. USA 86:9702 (1989), Lin et al., Biochemistry 28:1054 (1989), Pei et al., Proc. Natl. Acad. Sci. USA 87:9858 (1990), Strobel et al., Science 254:1639 (1991), and Posvic and Dervan, J. Am. Chem Soc. 112:9428 (1992)). Sequence specific DNA purification using triplex affinity capture has also been demonstrated. (Ito et al., Proc. Natl. Acad. Sci. USA 89:495 (1992)). Triplex forming oligonucleotides linked to intercalating agents such as acridine, or to cross-linking agents, such as p-azidophenacyl and psoralen, have been utilized, but only to enhance the stability of triplex binding. (Praseuth et al., Proc. Natl. Acad. Sci. USA 85:1349 (1988), Grigoriev et al., J. of Biological Chem. 267:3389 (1992), Takasugi et al., Proc. Natl. Acad. Sci. USA 88:5602 (1991).
A method for site-directed mutagenesis of a target DNA molecule would be a useful in achieving successful gene or anti-viral therapy. Such a method would also be a useful research tool for genetic engineering or for studying genetic mechanisms such as DNA repair.
Therefore, it is an object of the present invention to provide a method for in vivo and in vitro site-directed mutagenesis of a target DNA molecule.
It is a further object of the present invention to provide a method for mutagenesis of a target DNA molecule that is highly specific and efficient.
It is a further object of the present invention to provide a method for treating genetic disorders by gene therapy without the need for a viral vector.
It is a further object of the present invention to provide a method for treating cancer.
It is a further object of the present invention to provide a mutagenic oligonucleotide for use in therapy and research.